There exist various carotenoids in nature. Approximately 750 kinds of carotenoids have been identified heretofore, and many of the carotenoids have been shown to have useful physiological functions and industrial applicability such as an antioxidant activity or an antitumor activity, or a use as a functional pigment molecule. In general, carotenoids are compounds classified as tetraterpenes formed of an isoprene backbone of 30 or 40 carbon atoms. In nature, a linear backbone of 30 or 40 carbon atoms is formed as a basic backbone and then subjected to various modifications such as cyclization. Structural diversity of the carotenoids is attributed to diversity of such modifications. Further, carotenoids are known to have physiological activities greatly varying depending on structural differences based on the diversity of the modifications.
A large number of carotenoids have been obtained by isolation and extraction from nature such as plants, or by chemical synthesis. Recently, however, production utilizing microbial fermentation has also been performed. In order to establish biosynthetic pathways for rare carotenoids, which exist in nature in only trace amounts, and unnatural carotenoids, which do not exist in nature, research has been made by many researchers (Non Patent Literatures 1 to 8 and Patent Literature 1). In Non Patent Literatures 1 to 6, carotenoids having various structures have been obtained by synthesis based on the so-called combinatorial biosynthesis technique. The combinatorial biosynthesis is a technology involving altering biosynthetic pathways of microorganisms using a genetic engineering technique and allowing the microorganisms to produce a compound of interest. Modifying enzymes that provide various structures to carotenoids have the “locally specific” nature of recognizing and acting on only part of substrates. Based on such nature of the modified substrates, the combinatorial biosynthesis of the carotenoids has been performed (Non Patent Literature 9). Meanwhile, in Non Patent Literatures 7, 8, and 11 and Patent Literature 1, biosynthesis of various unnatural carotenoids has been achieved by a method involving constructing a metabolic pathway using an activity of an enzyme that cannot be found in nature and is created using protein engineering.
As described above, the carotenoids that exist in nature have backbones of 30 carbon atoms and 40 carbon atoms. The former is derived from 4,4′-diapophytoene, which is synthesized via head-to-head condensation of two molecules of farnesyl diphosphate (C15PP). The latter is derived from phytoene (carotenoid backbone compound of 40 carbon atoms), which is synthesized via head-to-head condensation of two molecules of geranylgeranyl diphosphate (C20PP). The former is a key component of biosynthetic pathways for 10-odd kinds of carotenoids known to exist in nature, and the latter is a key component of biosynthetic pathways for about 700 or more kinds of carotenoids.
Further, there is a report that a carotenoid having a backbone of 50 carbon atoms, which is larger than that of 40 carbon atoms, exists in nature (Patent Literature 2). The carotenoid having a backbone of 50 carbon atoms is synthesized by binding an isoprene unit to a carotenoid having 40 carbon atoms “as addition” so as to increase the total number of carbon atoms to 45 and 50 (Non Patent Literature 10). In a synthetic pathway for a backbone of 40 or more carbon atoms, such as a backbone of 50 carbon atoms or 60 carbon atoms, synthesis is performed by using, for example, geranylfarnesyl diphosphate (C25PP) or hexaprenyl diphosphate (C30PP) as a raw material. Although the carotenoid having a backbone of 40 or more carbon atoms is expected to have many potentialities for physiological and pigment functions and the like different from conventional ones, there is no detailed report on its synthetic pathways in nature, and there are few reports on its artificial biosynthetic pathways.
Dr. Umeno, one of the inventors of the present invention, developed an enzyme having a function of synthesizing a carotenoid backbone compound of 50 carbon atoms via condensation of two molecules of geranylfarnesyl diphosphate (C25PP) by altering a synthase (CrtM) for a carotenoid having 30 carbon atoms derived from Staphylococcus aureus. In addition, Dr. Umeno succeeded for the first time in the world in co-expressing the enzyme with an appropriate precursor synthase in Escherichia coli to produce 16,16′-diisopentenylphytoene, a carotenoid backbone compound of 50 carbon atoms (Non Patent Literature 7). However, in the synthetic pathway in Non Patent Literature 7, carotenoids having backbones of, for example, 30 carbon atoms, 40 carbon atoms, and 45 carbon atoms were synthesized simultaneously with that having a backbone of 50 carbon atoms. Even when a wild-type phytoene desaturase was added in this pathway, about 75% of the carotenoid backbone compound of 50 carbon atoms still remained without being desaturated, resulting in poor synthetic efficiency (Non Patent Literature 8).